It has been well over a decade since Cohen and Boyer first reported the use of bacterial plasmids as molecular cloning vectors; this marked the beginning of a new era in molecular biology and, historically, laid the foundation for what has been termed the "biotechnology industry." Cohen and Boyer's cloning vector, pSC101, relative to the cloning vectors in circulation today, seems almost quaint--insertion of foreign DNA fragments into pSC101 was limited to a single restriction enzyme cleavage site and to Escherichia coli as a host. During the past decade, the art has had access to hundreds of molecular cloning vectors having nearly as much applicability and diversity as the number of vectors. Irrespective of the variety of such vectors, the typical objective remains the same: increased availability of a protein of interest that ordinarily is produced naturally in minute quantities.
As noted, the advent of the biotechnology industry has allowed for the production of large quantifies of proteins. Proteins are the essential constituents of all living cells and proteins are comprised of combinations of 20 naturally occurring amino acids; each amino acid molecule is defined ("encoded") by groupings ("codons") of three deoxyribonucleic acid ("DNA") molecules; a string of DNA molecules ("DNA macromolecule") provides, in essence, a blueprint for the production of specific sequences of amino acids specified by that blueprint. Intimately involved in this process is ribonucleic acid ("RNA"); three types of RNA (messenger RNA; transfer RNA; and ribosomal RNA) convert the information encoded by the DNA into, eg a protein. Thus, genetic information is generally transferred as follows: DNA.fwdarw.RNA.fwdarw.protein.
In accordance with a typical strategy involving recombinant DNA technology, a DNA sequence which encodes a desired protein material ("cDNA") is identified and either isolated from a natural source or synthetically produced. By manipulating this piece of genetic material, the ends thereof are tailored to be ligated, or "fit," into a section of a small circular molecule of double stranded DNA. This circular molecule is typically referred to as a "DNA expression vector," or simply a "vector." The combination of the vector and the genetic material can be referred to as a "plasmid" and the plasmid can be replicated in a prokaryotic host (ie bacterial in nature) as an autonomous circular DNA molecule as the prokaryotic host replicates. Thereafter, the circular DNA plasmid can be isolated and introduced into a eukaryotic host (ie mammalian in nature) and host cells which have incorporated the plasmid DNA are selected. While some plasmid vectors will replicate as an autonomous circular DNA molecule in mammalian cells, (eg plasmids comprising Epstein Barr virus ("EBV") and Bovine Papilloma virus ("BPV") based vectors), most plasmids including DNA vectors, and all plasmids including RNA retroviral vectors, are integrated into the cellular DNA such that when the cellular DNA of the eukaryotic host cell replicates, the plasmid DNA will also replicate. Accordingly, as eukaryotic cells grow and divide, there is a corresponding increase in cells containing the integrated plasmid which leads to the production ("expression") of the protein material of interest. By subjecting the host cells containing the plasmid to favorable growth conditions, significant amounts of the host, and hence the protein of interest, are produced. Typically, the Chinese Hamster Ovary ("CHO") cell line is utilized as a eukaryotic host cell, while E. coli is utilized as a prokaryotic host cell.
The vector plays a crucial role in the foregoing--manipulation of the vector can allow for variability as to where the cDNA is inserted, means for determining whether the cDNA was, in fact, properly inserted within the vector, the conditions under which expression of the genetic material will or will not occur, etc. However, most of the vector manipulations are geared toward a single goal--increasing expression of a desired gene product, ie protein of interest. Stated again, most vector manipulation is conducted so that an "improved" vector will allow for production of a gene product at significantly higher levels when compared to a "non-improved" vector. Thus, while certain of the features/aspects/characteristics of one vector may appear to be similar to the features/aspects/characteristics of another vector, it is often necessary to examine the result of the overall goal of the manipulation--improved production of a gene product of interest.
While one "improved" vector may comprise characteristics which are desirable for one set of circumstances, these characteristics may not necessarily be desirable under other circumstances. However, one characteristic is desirable for all vectors: increased efficiency, ie the ability to increase the amount of protein of interest produced while at the same time decreasing the number of host cells to be screened which do not generate a sufficient amount of this protein. Such increased efficiency would have several desirable advantages, including reducing manufacturing costs and decreasing the time spent by technicians in screening for viable colonies which are expressing the protein of interest. Accordingly, what would be desirable and what would significantly improve the state of the art are expression vectors with such efficiency characteristics.